Radiation therapy plays a key role in pancreatic ductal adenocarcinoma (PDAC) treatment. However, it faces challenges from the fact that pancreatic cancer stem cells (PCSC) are resistant to ionizing radiation. Moreover, a recent finding indicates that radiation can reprogram nonstem- cancer cells into cancer stem cells, which is in agreement with our finding, namely iPCSC. Compelling evidence show that PDAC cells in expressing elevated levels of aldehyde dehydrogenase 1 (ALDH1), ALDHbrightcells, are PCSC/iPCSC. In the past, we demonstrated that cytotoxic T cells (CTL) specifically recognizing an epitope from the ALDH1A1 isoform eliminate ALDHbright cells in vitro and in vivo. However, the clinical applicability of adoptive T cell-based immunotherapy is limited by HLA class I restriction and requires ex vivo expansion of autologous or allogeneic T cells using a variety of methodologies. To overcome the limitations of CTL-targeting of ALDHbright cells, we tested the novel approach of using disulfiram (DSF) to target PCSC. DSF is a dithiocarbamate and an FDA-approve inhibitor of ALDH for treatment of alcoholism. Its toxicity to PDAC cells has been shown to be enhanced by the binding of the essential trace element copper (Cu++) to form DSF/Cu complexes. We found recently using PDAC cell lines that: i) PCSC increased two days after in vitro fractionated irradiation; ii) DSF/Cu depleted PCSC during the treatment gap, and abolished in vitro sphere formation of irradiated cells; iii) DSF/Cu downregulated Wnt pathway, a key cancer stem cell pathway, in irradiated PDAC cells, and iv) DSF/Cu blocked radiation induced re-expressing mRNA of stemness genes. These findings provide the rationale for our central hypothesis that DSF/Cu depletes pre-existing PCSC and radiation-induced PCSC which results in significant increases in the efficacy of radiation therapy. In Aim1, we will determine that DSF/Cu eliminates both PCSC and iPCSC in vitro and in vivo. In Aim2, we will examine that RT combined with DSF/Cu can effectively eliminate stem- and nonstem- PDAC cells in a neoadjuvant setting using xeno- and syngeneic- pancreatic tumors in mice. In Aim3, we will compare the efficacy of combination of DSF/Cu, RT and 5-Fluorouracil (5-FU), a commonly used radiosensitizer for PDAC vs conventional chemoradiation consisting of RT and 5-FU in a neoadjuvant setting in xeno- and syngeneic- PDAC mouse model systems . At the conclusion of the proposed studies, we will have evaluated DSF/Cu as a cytotoxic agent of pre-existing PSCS/iPSCS and as a blocker of generation of iPCSC, obtained knowledge of the mechanism(s) by which DSF/Cu enhances efficacy of radiation and chemoradiation therapy, and formed the foundation of a novel neoadjuvant therapy for improved efficacy of radiation and chemoradiation by combination with a FDA approved drug DSF and a nutrition supplement Copper gluconate.